To accurately diagnose various tissue diseases and conditions, medical personnel must remove one or more samples of tissue from the body of a patient. This process of harvesting tissue from the body is known as a biopsy. Once the tissue sample or samples are removed and sent to a pathology laboratory, the tissue will go through a series of procedures performed by a histotechnician and, ultimately, a pathologist, in order to diagnose one or more conditions associated with the tissue. The present invention generally relates to those procedures that are normally performed by the histotechnician to prepare the tissue sample or samples into slides that may be analyzed under a microscope by the pathologist.
Although the singular term “sample” is used throughout this specification, it should be understood that this term likewise encompasses plural “samples” as well. Once a tissue sample is removed from the body of a patient, it is typically placed into a specimen container containing a tissue fixative solution, such as formalin, and then the container is transported to a pathology laboratory. The tissue will undergo a process known as “grossing-in” in the pathology lab during which a histotechnician will retrieve the tissue sample from the container, typically cut the tissue into appropriate sizes for tissue processing, place individual samples into the appropriate sized small plastic tissue cassettes, and assign tracking numbers to each cassette. These tracking numbers are then logged into a tracking system used in the laboratory. For the smallest tissue samples, which may only be scrapings, the cassette includes fine mesh openings on the sides and bottoms so as not to lose the samples in the processor fluids. In other situations involving very small tissue samples, the samples are placed into a bag that resembles a tea bag that prevents the smallest tissue samples from escaping. Larger tissue samples are placed into cassettes having somewhat larger slotted openings which are nevertheless smaller than the tissue sample inside the cassette.
The cassettes are then placed into a stainless steel perforated basket and run through a tissue processing machine, often overnight. This machine uses a combination of vacuum, heat, and chemicals to remove the interstitial fluids within the tissue. Once the fluids have been removed from the tissue samples, the processing machine immerses the tissues samples in a bath of a hardenable material such as molten paraffin (i.e., a form of wax) so that the interstices in the tissue are replaced with paraffin. The histotechnician then removes the basket from the machine and removes the individual tissue cassettes. In a conventional procedure practiced for many years, the histotechnician individually removes the tissue sample from each cassette. The histotechnician must carefully orient the tissue sample, based on tissue type, into a stainless steel base mold that is roughly the size of the tissue cassette and is partially filled with molten paraffin. The tissue sample must be manually held, typically using forceps, flat against the bottom of the mold. If the tissue sample is not held flat against the bottom of the mold, this could compromise the ability to make proper slices of the tissue sample later in a microtome. Long thin tissue samples present a particular problem and must be held flat over their entire lengthwise surface so that the resulting slide will contain information indicative of the entire sample. The molten paraffin is then rapidly cooled on a refrigerated plate, which may be a thermal electric cooler (TEC), to partially solidify the paraffin thereby holding the tissue sample in the proper orientation against the bottom of the mold. The cassette is then placed on top of the base mold and an embedding material, which is also typically paraffin wax, is poured through the opened top of the cassette into the base mold. The cassette changes its function at this point in the procedure from a tissue holding component to a fixture type device for mounting in the microtome and making shavings or slices from the solidified paraffin and embedded tissue using the microtome. The base mold is chilled until all of the molten paraffin has hardened and the histotechnician removes the stainless steel base mold from the block of embedded paraffin. The tissue sample is thus embedded within a rectangular block of hard paraffin with a plastic tissue cassette on the opposite side. As mentioned, the cassette may then be used as a holder or fixture in the chuck of the microtome. As with the tissue processing machine, the embedding process is accomplished in a batch fashion during which an average histotechnician may embed approximately 40 to 60 cassettes per hour.
The blocks of hardened paraffin containing the embedded tissue samples are then ready to be sliced into extremely thin sections for placement on a microscope slide. The histotechnician mounts the embedded tissue block in a chuck on the microtome that is sized to accept the side of the block that has the embedded plastic cassette. The histotechnician can then begin slicing the paraffin block which has the tissue sample embedded opposite to the plastic cassette surface. This yields a ribbon of individual slices of the tissue embedded in the hardened paraffin. The action of the microtome causes the individual slices to stick together when done properly and, subsequently, these very thin ribbons of slices are floated into a water bath and a glass slide is carefully placed underneath the slice. The slice, with the thin sectioned tissue sample embedded therein, is then adhered to the top of the slide.
When the histotechnician has enough slides from the tissue sample, the slides are placed into an automatic staining machine. The staining machine goes through a series of infiltrating steps to stain the different tissue and cells of the slide different colors. This helps the pathologist identify different structures and makes it easier to find any abnormalities in the tissue. After the staining procedure is complete, the slides are cover slipped and prepared for the pathologist to place under a microscope for analysis.
Based on the summary of the procedure provided above, it will be appreciated that conventional tissue sample handling and processing is a very labor-intensive process involving several manual steps performed by a histotechnician. Thus, repetitive stress injuries such as carpal tunnel syndrome are prevalent. This is especially true with the tissue sample embedding process. These multiple manual operations and repeated tissue handling increase the likelihood of human error and, moreover, require highly trained and skilled histotechnicians to ensure that the tissue samples ultimately adhered to the slides for analysis by the pathologist are in an optimum condition and orientation to make accurate diagnoses.
U.S. Pat. Nos. 5,817,032 (the '032 patent) and 7,156,814, and U.S. Patent Application Publication Nos. 2005/0226770; 2005/0147538; and 2005/0084425 disclose various improvements to this area of technology, including new manners of holding tissue samples during the grossing in, embedding, and microtome or slicing procedures. The disclosures of (the '032 patent) and U.S. Patent Application Publication Nos. 2005/0226770; 2005/0147538; and 2005/0084425 are hereby fully incorporated by reference herein. For example, the '032 patent relates to a tissue trapping and supporting device, which may be a cassette, and which may be successfully sectioned using a microtome. When such a sectionable cassette is used, the tissue sample is immobilized within the cassette and subjected to the process for replacing tissue fluids with paraffin. Then, the tissue sample and the cassette are sliced at the same time for later mounting on microscope slides. Because the tissue sample is never removed from the cassette from the time it is processed in the tissue processing machine to the time that it is cut or sliced with the microtome, a significant amount of handling time is saved. Moreover, the chance for human error or tissue loss is significantly reduced due to the elimination of separate tissue handling steps. The '032 patent and the above-incorporated published applications also generally disclose further improvements that help to automate the overall process and, in conjunction with the novel tissue supports (e.g., cassettes), can even further reduce the handling steps during the entire procedure and make the procedure more reliable.
Sectionable cassettes for histopathology, such as mentioned above, need to accommodate many different types of tissue. It is up to the histopathology technician to orient the tissue for processing and paraffin embedding so as to guarantee the availability of optimum diagnostic information from the eventual microscopic slide made from sections of the processed tissue. In some cases, tissue samples do not require any delicate or specific orientation for sectioning. Other tissue types require very specific orientation during the embedding process.
Standard practices for tissue orientation and embedding techniques are well known and understood in the art. The use of sectionable cassettes makes changes to some of these standard practices necessary and makes the need for more tools and devices to aid the process evident. In the process, such as that previously disclosed for sectionable cassettes, final orientation and alignment of tissues in the paraffin block are determined prior to closing the lid of the cassette and sending it through the processor. There is no opportunity for reorientation prior to paraffin embedding. This is one of the most important benefits to the automation process as the tissue is handled only once as it is placed into a cassette with no further downstream human intervention. This necessitates that the initial tissue orientation and placement in the cassette be correct and not subject to change during the process. While large samples require only moderate attention to the precision of tissue orientation, the opposite is true for small biopsies such as those produced during dermatology procedures. The pathology lab is a busy environment and throughput of tissue samples must be maintained to keep up with the case load. Automation of the histopathology lab is being implemented on a regular basis. While automation is being embraced, the inventors of this technology have had to continually make improvements and innovations to insure that the quality of the slides is not compromised by the introduction of the automation steps or devices. Any steps introduced must be cost effective and time-efficient. Therefore, the need to reduce time and steps in the process is mandatory, while the quality of the tissue sample preparation and diagnostic slide must remain extremely high.
In order for automation in the histopathology lab to be widely accepted it is imperative that all types of tissues can be embedded in sections correctly. One of the most challenging types of tissue to properly orient and embed is that of skin samples. With the increase of skin cancers all around the world, the number of corresponding diagnostic procedures that must be carried out on the biopsy samples created from skin lesion removals has increased as well. Once a skin lesion is identified for removal many different types of surgical intervention can be undertaken. One of the simplest and fastest removals is a shave biopsy. To perform a shave biopsy the lesion is first pinched by the surgeon, between his thumb and forefinger. He then takes a shallow shave cut parallel to the skin to remove the lesion. This is usually done on lesions that are less than 6 mm in diameter and in most cases only removes the top layers of the skin. Most doctors will send all biopsies to the pathology lab to confirm that there are no cancerous cells in the removed tissue. Because shave biopsies are thin and small, they are particularly difficult to orient properly in the paraffin for tissue sections. When embedded by hand, small forceps must be used to hold the thin tissue samples against the bottom of a metal paraffin mold with the proper orientation until the paraffin has cooled. It can then be difficult to remove the forceps without dislodging the tissue sample and altering its orientation. This procedure must be repeated so that all of the tissue samples from that procedure are properly embedded in the same block. If the paraffin has solidified enough to retain the first sample, it may be too solidified to accept the second sample. This sets in motion a battle between the paraffin being either too hot or too cold and the histopathology technician struggling to see through the now opaque and rapidly solidifying paraffin, trying to make sure that the second sample is properly oriented. This procedure is delicate and tedious and often produces less than optimal results.
While the shave biopsy method may be an effective and quick lesion removal for benign lesions it is not appropriate if one suspects the presence of cancerous cells. In those cases a more invasive procedure using a biopsy punch or surgical scalpel is required to remove the lesion down through the full dermis to the fat layer. This produces a thicker tissue sample which again must be properly oriented during the embedding process to create the proper diagnostic slide. For a skin biopsy to be properly oriented for diagnostic review the gross in process and embedding procedure is typically as follows. The tissue sample is removed from the biopsy transport and fixation container. The formalin solution in the biopsy container usually causes the biopsy sample to curl and become distorted. The histopathology technician must first remove the tissue from the sample container and attempt to gently flatten it on the cutting board so that the lesion can be viewed as it appeared on the patient. The tissue sample should then be transected through the lesion. The two exposed freshly cut edges will then need to be oriented parallel to the eventual microtome sectioning surface. This gives the pathologist the correct diagnostic cross-section of the lesion in which to stage any disease. It is important to understand that when diagnosing skin disease the depth of the intrusion of the disease is far more important as a measure of its invasiveness than is the diameter on the surface of the skin. That is why it is so important to have the proper cross-section view of each lesion to be able to assess how far into the skin layers cancer cells have penetrated. Therefore, any device which is intended to enhance or enable the tissue orientation process for automated embedding must preserve these very specific orientation requirements in order to be useful.
With specific regard to prior art devices designed by this inventor, novel improvements have been made from disclosures in U.S. Pat. No. 7,156,814 which render far superior results in the case of tissue handling and orientation, tissue processing, paraffin embedding, microtome sectioning, slide preparation and, finally, diagnostic usefulness.
Note that while embodiments in this disclosure are primarily directed toward tissue orientation and holding alignment devices for use with sectionable tissue embedding cassettes and automated systems, the orientation device itself can be manually embedded in a paraffin block and sectioned along with the tissue without the use of an automated embedding machine or process. Therefore, no limitation should be construed upon the orientation device for use only with sectionable cassettes.
Small, thin samples like those derived from skin shave biopsies or needle biopsies are too thin and elongated to be held in place by a single pincher point. The tissue needs to be supported along its longest axis to prevent it from curling and pulling away from the sectioning plane. In the prior art it was very difficult to orient and retain the samples all on the same sectioning plane.
One of the challenges encountered with devices in the '814 patent, such as those disclosed in FIGS. 78a-80, is the procedure for gripping the tissue prior to its placement in a cassette. The '814 disclosure taught holding the tissue on edge and placing the orientation device over top of it. This proved to be problematic in situations where the tissue has no intrinsic strength to hold its own shape. It is also a problem when the tissue samples are extremely small and human dexterity is challenged working with those sizes. Other important hurdles to overcome include the potential for of crush artifact in the tissue, which can impede diagnosis, and the potential for air bubbles in the embedding material, which can impede high quality ribbon slices. In the past, these concerns have competed with the goal of accurately orienting and retaining the original presentation and orientation of the tissue sample throughout processing and embedding. Clearly, a more user-friendly solution is needed.
In spite of the various advances made in this field, there is an increasing need for additional improvements related to increased production capability and more consistent quality of embedded tissue samples and resulting slices or ribbons of embedded tissue that will be subject to diagnosis. This can be especially important when handling smaller tissue sample sizes, such as very small elongated tissue samples produced from coring or needle biopsy instruments. In addition some tissue samples like skin lesion biopsies must maintain special orientation throughout tissue processing and embedding steps. Although the improvements to be disclosed herein are applicable to any tissue sample sizes, there are specific biopsy samples which regardless of their size must maintain either proper orientation or extreme flatness to allow for diagnostically correct sections to be obtained for the production of microscope slides. Core biopsy tissue samples present a particular challenge in keeping the entire core sample held flat along its entire length in the tissue supporting structure. This challenge is exacerbated by the actual core sample harvesting process. Currently, the practitioner, most often a radiologist, performs the core biopsy procedure with a needle. After harvesting, the core sample is ejected from the needle directly into a bottle containing a solution of buffered formaldehyde or formalin. This solution “fixes” the tissue. The fixing process preserves the tissue and prevents degradation and contamination, but also hardens the tissue and causes the tissue to curl up as it contracts due to the interaction with the formalin. This curl artifact is highly undesirable because the tissue needs to be held flat while it is embedded in the paraffin. There is a significant need for improvements designed to straighten and hold the core sample flat during the embedding process.